Abstract

Lipid modification of cytoplasmic proteins initiates membrane engagement that triggers diverse cellular processes. Despite the abundance of lipidated proteins in the human proteome, the key determinants underlying membrane recognition and insertion are poorly understood. Here, we define the course of spontaneous membrane insertion of LC3 protein modified with phosphatidylethanolamine using multiple coarse-grain simulations. The partitioning of the lipid anchor chains proceeds through a concerted process, with its two acyl chains inserting one after the other. Concurrently, a conformational rearrangement involving the α-helix III of LC3, especially in the three basic residues Lys65, Arg68, and Arg69, ensures stable insertion of the phosphatidylethanolamine anchor into membranes. Mutational studies validate the crucial role of these residues, and further live-cell imaging analysis shows a substantial reduction in the formation of autophagic vesicles for the mutant proteins. Our study captures the process of water-favored LC3 protein recruitment to the membrane and thus opens, to our knowledge, new avenues to explore the cellular dynamics underlying vesicular trafficking.

Structural properties of LC3-PE and its spontaneous membrane insertion. (a) The structure of the LC3 protein shown in CG representation, with the PE chain covalently attached to its C-terminus. The secondary structural elements are comprised of four α-helices and five β-sheets shown in green. (b) A schematic representation of the LC3-PE protein linked with the lipid anchor chain (blue) and Gly120 at the C-terminus (red) highlighted for clarity. (c) Amino acid alignment of LC3 with its homolog yeast protein Atg8, showing the conserved Gly120 residue. (d) Time evolution of the distance between the PE chain of LC3 and the POPC bilayer along the 15 trajectories. The protein inserts in trajectories 1–8 and 10–15. In trajectory 9, the PE chain insertion is not observed. (e) Snapshots of LC3-PE insertion. The structures were extracted from trajectory 3, one of the representative productive trajectories. The LC3-PE transition from an aqueous state (water not shown for clarity) to the membrane-bound and finally to the membrane-inserted state is shown. To see this figure in color, go online.

Localization of protein onto the membrane. (a) The residue-wise distribution of the distance of the protein from the membrane. The secondary structural elements are mentioned at the top. The highlighted box shows the structural interface that makes close contact with the membrane in all simulations. Error bars show the standard error calculated for the period of the trajectory subsequent to insertion, for all simulations combined. In addition, see for the preinsertion-phase residue-wise distribution. (b) LC3 displays a remarkably asymmetric surface charge distribution with a contiguous segment of positively charged surface (dark blue) and the α-helix III region highlighted in light blue. (c) Time evolution of the distance of α-helix III, β-sheet IV, and β-sheet V from the membrane in one of the representative productive simulations. For details of the time kinetics of individual acyl chain insertion events, see . (d) The probability of contact formation of α-helix III residues with the membrane. Data on the left are calculated from all productive LC3-PE simulations of LC3 after insertion and those on the right from the single nonproductive trajectory 9. The error bars shown represent the mean ± SE of all simulations. A distance cutoff of 0.8 nm between each protein residue and the membrane was used. To see this figure in color, go online.

In silico mutations reveal altered protein-membrane interactions. (a) Snapshots displaying the mutated residues at the pre- and postinsertion states of the LC3 protein. (b and c) The time course of the Z-component of the center of mass of the membrane and the PE chain of Ala- and Ile-mutant proteins, respectively The distance color bar is shown on the right, with the dark blue color indicating membrane insertion of the lipid anchor. To see this figure in color, go online.

Experimental validation of how mutation of residues crucial to membrane targeting affects autophagosome formation. (a) Immunofluorescence micrographs of HEK cells cotransfected with mCherry-LC3 mutants along with wild-type LC3 and a vector control. The formation of punctate structures was notably reduced in the LC3-K65A-R68A-R69E mutant. Scale bars, 5μm. (b) Quantitation of the number of puncta in cells transfected with wild-type LC3 and mutants using Volocity software ( from three independent experiments). Bars represent the mean ± SD across replicates. ∗p ≤ 0.05; ∗∗p ≤ 0.01; and ∗∗∗p ≤ 0.001. (c) Western blot analysis for monitoring LC3 flux. HEK cell lysates expressing mCherry-LC3 and LC3 mutants were subjected to immunoblotting with anti-mCherry and anti-β-actin antibodies. The presence of both nonlipidated and lipidated LC3 was observed. To see this figure in color, go online.

Role of electrostatistics in protein-membrane interaction. (A) Time evolution of the distance between the PE chain of the LC3 and the negatively charged bilayer along the 15 trajectories. The lipid anchor of the protein inserts in all trajectories. (B) Localization of the protein during four representative trajectories. The bars represent the existence of WA (maroon), MA (yellow), and MI (dark blue) states. See Materials and Methods for details regarding the definition of the different parameters. To see this figure in color, go online.

The nonlipidated protein. Shown is a time series of the distance between the nonlipidated LC3 protein and the center of mass of the membrane across five independent simulations. The protein tends to be in the aqueous phase (distance ≥ 6 nm), with fast-timescale membrane dissociation events. The color gradient reflects the distance (in nanometers) for the MA (green) and WA (orange/red) states. To see this figure in color, go online.

Pressure profiles. Comparison of the lateral pressure profiles of the productive simulations (black) and the pure POPC bilayer without protein (red) as control simulations. The pressure profiles were calculated after the insertion of the lipid anchor for the productive simulations. The error bars shown represent the standard error between the simulations. For further details, see Materials and Methods. To see this figure in color, go online.

Schematic representation of the mechanism of lipidated-LC3 membrane insertion. The left part of the diagram shows an unproductive insertion event, with the protein’s hydrophobic anchor not inserted into the membrane. Both the lipid anchor (control simulations) and key residues in the protein (experimental mutations) are necessary for insertion of the lipidated protein. These dynamic changes within the lipidated protein are induced by specific protein-lipid interactions. This specificity determines and facilitates PE insertion, as shown at the right of the diagram. To see this figure in color, go online.